Despite the use of RNA structure as a model for in silico evolution, and extensive in vitro study of RNA evolution, the forces contributing to the genesis, adaptation, and maintenance of structured RNA regulators in the context of an organism are largely unexplored. In many bacteria, structured RNAs regulate processes that are essential for viability or virulence. Due to their association with these processes and complex three- dimensional structures, several such RNA regulators are targets of antibiotic development. Like protein targets of antibiotics, one mechanism for resistance is modification of ligand specificity or inactivation of the RNA regulator. Understanding the roles that structured RNA regulators play in bacterial fitness and virulence, and the ways in which bacteria adapt to their loss is key to preserving structured RNA regulators as viable antibacterial targets. The goal of this project is to assess the fitness cost of inactivating structured RNA regulators in the gram positive bacteria Bacillus subtilis and Streptococcus pneumoniae, and to determine how organisms compensate for the loss of regulation by altering either the original locus, or other genomic regions. Of the 10 structured RNA cis-regulators shared between B. subtilis and S. pneumoniae, many regulate genes that are instrumental to virulence or viability. The central hypothesis is that some inactivated RNA regulators will have a larger influence on organismal fitness and virulence than others due to their impact on global metabolic flux or assembly of macromolecular complexes. The first aim measures the fitness cost conferred by ligand-insensitive RNA regulators in the context of both defined culture medium conditions and a mouse infection model. The second aim determines the extent that other cellular processes are impacted by the loss of regulation. To assess this, global gene expression changes and genetic interactions for each of the RNA regulators will be mapped using transcriptomics and genome-wide transposon mutagenesis coupled with high- throughput sequencing (Tn-seq). The third aim establishes how an organism adapts to loss of an RNA regulator using experimental evolution. Preliminary results indicate that the locus of the RNA regulator may be under strong selective pressure, providing a window into how RNA structures evolve and adapt within the context of organism. By combining the extensive mechanistic knowledge of RNA regulators in B. subtilis and the S. pneumoniae model for infection with high-throughput approaches to assess genomic changes and their impacts on metabolism, this work explores a fundamental biological question to derive conclusions that will inform antimicrobial development.